Dispase 2

Fresh tumor tissues or spleens were mechanically dissociated to minced meat and digested for 30 min at 37°C in RPMI 1640 with 2 mg/ml Collagenase IV (17104-019, Gibco), 2000 U/ml DNase I (D7073, Beyotime Biotechnology), 0.5 mg/ml Hyaluronidase (S10060, YuanYe Biotechnology) and 0.5 mg/ml Dispase II (S25046, YuanYe Biotechnology). 20–30 ml of 1 × PBS containing 2% FBS (F-PBS) was added to stop the digestive reaction. Subsequently, red blood cell lysis was used to remove red blood cells for 5 min, and the lysis reaction then stopped by adding 20–30 ml of F-PBS. Then, the cell suspension was filtrated on a 70-μm cell strainer (15-1070, BIOLOGIX) and 40-μm cell strainer (15-1040, BIOLOGIX), and washed with F-PBS. In this way, a single cell suspension of the tumor tissue or spleen was obtained. Finally, the cells were counted and tested for viability by Countess^TM automated cell counter (C10281, Invitrogen) to wait for the next step.

Peritumour tissue and ccRCC tissue from ccRCC patients were individually dissociated in 50 U/mml of Dispase II (S25046, YuanyeBio-Technology, China) on gentleMACS octo with Heater (Miltenyi Biotec, German) to enable efficient dissociation of lymphocytes from the tumour cells and stromal tissue. Staining steps were performed at 4 ℃ over 20 min with FACS buffer washes between steps. Flow cytometry antibodies were used: FITC-CD3 (Cat: 317305, Clone: OKT3, 1:20, Biolegend), PE-IFN-γ (Cat: 502508, Clone: 4SB3, 1:20, Biolegend), APC-PD-1 (Cat: 379207, Clone: A17188A, 1:20, Biolegend).